In the progress of arthritis, synovium, cartilage, and bone are all sites of increased production of growth factors, cytokines, and inflammatory mediators that are believed to contribute to pathogenesis [1, 2]. Although both bone and synovium have important roles in the pathogenesis of arthritis [1,3], most efforts at developing disease-modifying treatments have focused on the molecular events within cartilage. Arthritic chondrocytes undergo a series of complex changes, including proliferation, catabolic alteration, and, ultimately, death. The regulation of these phenotypic changes at different stages of disease is under intensive study, with focus on the biomechanical and biochemical signals that regulate each of these discrete chondrocyte responses [2, 4]. Chondrocytes themselves are major protagonists in this regulatory cascade—not just the target of external biomechanical and biochemical stimuli, but themselves the source of cytokines, proteases, and inflammatory mediators that promote the deterioration of articular cartilage [1, 2]. Pathogenic molecules produced by arthritic chondrocytes include tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, IL-8, matrix metalloproteinases (MMPs), ADAMTSs, nitric oxide, prostaglandins, and leukotrienes [2, 4]. There is also evidence that arthritic chondrocytes exhibit increased anabolic activity, including increased release of growth factors and synthesis of type II collagen, proteoglycan, and other extracellular matrix proteins, as well as the expression of genes associated with the chondroprogenitor hypertrophic phenotype [5-7].
A great deal of research in rheumatology over the past two decades has focused on identifying cytokines and mediators responsible for the inflammatory and degenerative processes in rheumatoid arthritis (RA), with the aim of developing specific antagonists of therapeutic value. Among all factors, TNF-a has received the greatest attention because of its position at the apex of the pro-inflammatory cytokine cascade, and its dominance in the pathogenesis of RA. Many lines of evidence support this theory including: (1) TNF-a is expressed at high levels in inflamed synovium and cartilage from RA patients; (2) anti-TNF-a inhibits the production of other pro-inflammatory cytokines including IL-1; and (3) TNF-a can induce joint inflammation, trigger cartilage destruction by inducing metalloproteinase, and stimulate osteoclastogenesis and bone resorption. Most importantly, anti-TNF therapies for RA have shown remarkable results by decreasing inflammation, improving patient function and vitality, and attenuating cartilage and bone erosions. There are now three anti-TNF treatments via targeting to TNF ligand, etanercept (Enbrel, a soluble TNFR2-IgG1 fusion protein), infliximab (Remicade, a chimeric monoclonal antibody against TNF-a), and adalimumab (a humaneric monoclonal antibody against TNF-a) that have been used clinically for treating various kinds of inflammatory diseases, including rheumatoid arthritis. Engineered proteins/peptides are now providing a new wave of therapeutic products. Indeed, designed protein/peptide therapeutic agents now outnumber and surpass the number of new small-molecule drugs approved annually by the FDA. Antibodies and immunoadhesins that directly target cytokines for their systemic removal (ligand ablation) have become an effective therapeutic strategy (e.g. etanercept, adalimumab and infliximab), and in some indications the selective targeting of cytokine receptors (e.g. anakinra) can deliver a highly effective clinical outcome.
Granulin/epithelin precursor (GEP), also known as PC-cell-derived growth factor (PCDGF), acrogranin, progranulin (PGRN), proepithelin (PEPI), or GP80, was first purified as a growth factor from conditioned tissue culture media [8, 9, 65, 66, 67]. GEP is a 593-amino-acid secreted glycoprotein with an apparent molecular weight of 80 kDa [10, 14], which acts as an autocrine growth factor. GEP contains seven and a half repeats of a cysteine-rich motif (CX5-6CX5CCX8CCX6CCXDX2HCCPX4CX5-6C) (SEQ ID NO: 9) in the order P-G-F-B-A-C-D-E, where A-G are full repeats and P is the half motif (FIG. 1). The C-terminal region of the consensus sequence contains the conserved sequence CCXDX2HCCP (SEQ ID NO: 10) and is suggested to have a metal binding site and to be involved in regulatory function [15]. Notably, GEP undergoes proteolytic processing with the liberation of small, 6-kDa repeat units known as granulins (or epithelins), which retain biological activity [16]: peptides are active in cell growth assays [13] and may be related to inflammation [17].
GEP is abundantly expressed in rapidly cycling epithelial cells, in cells of the immune system, and in neurons [10-12, 17]. High levels of GEP expression are also found in several human cancers and contribute to tumorigenesis in diverse cancers, including breast cancer, clear cell renal carcinoma, invasive ovarian carcinoma, glioblastoma, adipocytic teratoma, and multiple myeloma [16, 18-24]. Although GEP mainly functions as a secreted growth factor, it was also found to be localized inside cells and to directly modulate intracellular activities [12, 25-27]. The role of GEP in the regulation of cellular proliferation has been well characterized using mouse embryo fibroblasts derived from mice with a targeted deletion of the insulin-like growth factor receptor (IGF-IR) gene (R-cells). These cells are unable to proliferate in response to IGF-1 and other growth factors (EGF and PDGF) necessary for progression through the cell cycle [28]. In contrast, GEP is the only known growth factor able to bypass the requirement for the IGF-IR, thus promoting cell growth of R-cells [13, 29]. Increasing evidence has also implicated GEP in the regulation of differentiation, development and pathological processes. It has been isolated as a differentially-expressed gene from mesothelial differentiation [30], sexual differentiation of the brain [31], macrophage development [32], and synovium of rheumatoid arthritis and osteoarthritis [33]. GEP was also shown to be a crucial mediator of wound response and tissue repair [21, 34]. It was reported that mutations in GEP cause tau-negative frontotemporal dementia linked to chromosome 17 [35-38]. The mode of action of GEP remains largely unknown. Several GEP-associated proteins have been reported to affect GEP action in various processes. One example is the secretory leukocyte protease inhibitor (SLPI). Elastase digests GEP exclusively in the intergranulin linkers, resulting in the generation of granulin peptides, suggesting that this protease may be an important GEP convertase. SLPI blocks this proteolysis either by directly binding to elastase or by sequestering granulin peptides from the enzyme [34]. GEP can modulate transcriptional activities by interacting with human cyclin T1 [26] and Tat-P-TEFb [25]. GEP was also found to interact with perlecan, a heparan sulfate proteoglycan; perlecan-null mice exhibit severe skeletal defects [19, 39-41].
The Tumor Necrosis Factor (TNF) family of cytokines plays an essential role in multiple biological functions including inflammation, organogenesis, host defense, autoimmunity, and apoptosis. The action of these potent biological mediators is achieved through a receptor-ligand interaction, leading to intracellular signaling and a change in cellular phenotype. The ligands exert their function by forming trimers and binding to their corresponding receptors. Subsequent receptor oligomerization results in conformational change of the receptor's intracellular domain, which then allows for members of the TNF receptor-associated factor (TRAF) family of adaptor proteins to bind and initiate a signaling cascade. TNFR2, TNFR1, TrkA, NGFR, CD 40, CD 30, OX-40, DR5, DR3, DR4 and RANK include some of the members of TNF receptor super-family that interact with different TRAF molecules (including 1-6) (Lewit-Bentley, A., et al., J. Mol. Biol. 199:389-392 (1988), Banner, D. W., et al., Cel. 73:431-445 (1993), Karpusas, M., et al., Structure. 3:1031-1039 (1995), Hymowitz, S. G., et al., Mol. Cell. 4:563-571 (1999), Mongkilsapaya, J., et al., Nat. Struc. Biol. 6:-1048-1053 (1999), Cha, S. S., et al., J. Biol. Chem. 275:31171-31177 (2000)).
Both TNF receptors (TNFR1 and TNFR2) are ubiquitously expressed in cells and interact with their cognate ligand: TNFα, a central proinflammatory cytokine [42-44]. It is widely accepted that TNFα serves very important functions in pathophysiology, being a factor that interferes strongly with the cell growth, differentiation and death. TNF appears not only to orchestrate acute responses to infection and immunological injury but also to act as a balancing factor required for the re-establishment of physiological homeostasis and regulation [45]. TNFα has been found to affect skeletal development: its level is increased in most inflammatory diseases known to affect longitudinal growth in children [46, 47] and catch-up growth was shown in children with refractory juvenile idiopathic arthritis treated with the TNF antagonist etanercept (Enbrel) [46, 47]; TNFα regulates growth plate chondrocytes and suppress longitudinal growth in metatarsal organ cultures [48].
Arthritis is a degenerative joint disease, occurring primarily in the senior population, that currently affects more than 46,000,000 individuals in the United States. Typical clinical symptoms are pain and stiffness, particularly after prolonged activity. In industrialized societies arthritis is the leading cause of physical disability, increased health care usage, and impaired quality of life. The impact of arthritic conditions is expected to grow as the population both increases and ages in the coming decades. Despite the prevalence of arthritic diseases, their precise etiology, pathogenesis, and progression remain beyond our understanding. Evidence is accumulating that demonstrates the significance of inflammatory cytokines and growth factors in the pathological processes of arthritis. The destruction of the extracellular matrices of articular cartilage and bone in arthritic joints is thought to be mediated by excessive cytokine activities and imbalance between inflammatory cytokines and their physiological antagonists. The isolation of the growth factors that regulates chondrocytes and arthritis, and the inhibitors that antagonize the actions of cytokines, are therefore of great importance from both a pathophysiological and a therapeutic standpoint. We have previously identified granulin/epithelin precursor (GEP) as a novel chondrogenic growth factor that plays an essential role in cartilage formation (Xu, K et al (2007) J Biol Chem 282(15):11347-11355; WO 2008/094687 A2).
There still exists a need in the art for a better and more complete understanding of the process of and, thereby, intervention for, inflammatory diseases and conditions, particularly TNF family member mediated processes and conditions. Thus, the purpose of this invention is to extend our understanding of the molecular mechanisms by which growth factors and cytokines control cartilage development and arthritis, and to mediators thereof for development of new anti-TNF/TNFR therapeutic interventions for various kinds of TNF-related diseases, including inflammatory arthritis.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.